Competition of Charge and Energy Transfer Processes in Donor-Acceptor Fluorescence Pairs: Calibrating the Spectroscopic Ruler

Sensing strategies utilizing Forster resonance energy transfer (FRET) are widely used for probing biological phenomena. FRET sensitivity to the donor acceptor distance makes it ideal for measuring the concentration of a known analyte or determining the spatial separation between fluorescent labels in a macro molecular assembly. The difficulty lies in extracting the FRET efficiency from the acceptor-induced quenching of the donor emission, which may contain a significant non FRET contribution. Here, we demonstrate a general spectroscopic approach for differentiating between charge transfer and energy transfer (ET) processes in donor-acceptor assemblies and apply the developed method for unravelling the FRET/non-FRET contributions in cyanine dye-semiconductor quantum dot (QD) constructs. The present method relies on correlating the amplitude of the acceptor emission to specific changes in the donor excitation profile in order to extract ET-only transfer efficiencies. Quenching of the donor emission is then utilized to determine the non-ET component, tentatively attributed to the charge transfer. We observe that the latter accounts for 50-99% of donor emission quenching in QD-Cy5 and QD-Cy7 systems, stressing the importance of determining the non-FRET efficiency in a spectroscopic ruler and other FRET-based sensing applications.